Diarrhea is one of the most frequent complaints in deployed military personnel from the United States and has significant adverse effects on the health of service members. It was reported that 76.8 % of soldiers in Operation Iraqi Freedom and Operation Enduring Freedom experienced diarrhea. Diabetes is becoming an increasing health concern for veterans, with one in four veterans receiving care from the Department of Veterans Affairs has diabetes. A common troublesome gastrointestinal complication of diabetes is diarrhea. Diabetic diarrhea attains clinical significance because of its severity and refractory nature. The overall incidence of diabetic diarrhea can reach as high as 22%. Although diarrhea is less frequent in type 2 diabetic mellitus (T2DM), the frequent cause of diarrhea in T2DM is associated with drugs, including metformin, which is commonly used for glycemic control in T2DM. Clinical Relevance to the Department of Veterans Affair is that with more than 70% of patients in VA facilities being overweight or obese, T2DM is a major health concern. Yet the underlying cause of diarrhea in T2DM has not been studied and there is a need to improve treatment for diabetic diarrhea. Diarrhea is caused by altered intestinal transport of electrolytes and fluid, but the link between the aberrant electrolyte transport and diabetic diarrhea is not established. The major Na+ absorptive mechanism in the intestine is electroneutral NaCl absorption mediated by the Na+/H+ exchanger 3 (NHE3). Inhibition of NHE3 is associated with both enterotoxigenic and inflammatory diarrhea. Our recent study of type 1 diabetic mellitus (T1DM) showed that NHE3 expression is downregulated in T1DM humans and mice, which helped to identify a specific ion transporter as a cause of diabetic diarrhea for the first time. Preliminary studies have demonstrated that NHE3 expression is decreased in human diabetic tissues and db/db mice, a mouse model for T2DM. Additionally, we have compelling evidence that metformin, a widely prescribed drug to treat T2DM, inhibits NHE3, suggesting NHE3 dysfunction is associated with frequent diarrhea caused by metformin. Adenosine monophosphate kinase (AMPK) is a major effector of anti-diabetic metformin, and activation of AMPK causes NHE3 inhibition, suggesting the critical role of AMPK in NHE3 regulation by metformin. The objective of the proposed study is determine the impact of decreased NHE3 expression and activity in diabetic diarrhea, in particular T2DM. The central hypothesis of this proposed study is that inhibition of NHE3 by elevated PKC? is a major cause of NHE3 inhibition in T2DM, and activation of AMPK by anti-diabetic drugs such as metformin further inhibits NHE3, contributing to diarrhea in some patients. The proposed studies designed to test this hypothesis will establish a new paradigm that diabetic diarrhea is caused by aberrant regulation of sodium and fluid transport by NHE3. We propose to test the hypothesis that PKC? is a major cause of reduced NHE3 activity and fluid absorption in T2DM mice using intestinal epithelial cells and experimental models of T2DM (Aim 1). We propose to determine that AMPK inhibits NHE3 in vitro and in vivo. We will determine the underlying mechanism of NHE3 inhibition by AMPK by investigating signal pathways responsible NHE3 inhibition. We also plan to determine whether AMPK activation by metformin mediates NHE3 inhibition via ubiquitination of NHE3 by using humanized mice (Aim 2). As a pre-clinical test to improve the treatment for diabetic diarrhea, we will test the efficacy of a biologically occurring phospholipid, lysophosphatidic acid, and probiotics in mitigating the inhibition of NHE3 (Aim 3).